Rapid determination of baicalin and total baicalein content in Scutellariae radix by ATR-IR and NIR spectroscopy.

In this study methods for the quantification of baicalin and total baicalein in Scutellariae radix with near infrared (NIR) spectroscopy and attenuated-total-reflectance mid-infrared (ATR-IR) spectroscopy in hyphenation with multivariate analysis were developed and compared. The reference analysis was performed by high performance liquid chromatography coupled to diode array detection (HPLC-DAD). Different pretreatments like standard normal variate (SNV), multiplicative scatter correction (MSC), first and second derivative Savitzky-Golay were applied on the spectra to optimize the calibrations. A principal component analysis was performed with both spectroscopic methods to distinguish wild and cultivated samples. Quality parameters obtained for test-set calibration models of ATR-IR spectroscopy (baicalin: standard error of prediction (SEP)=1.31, ratio performance to deviation (RPD)=2.91 and R(2)=0.88; total baicalein: SEP=1.02, RPD=3.24 and R(2)=0.89) and NIR spectroscopy (baicalin: SEP=1.50, RPD=2.54 and R(2)=0.88; total baicalein: SEP=1.19, RPD=2.76 and R(2)=0.84) demonstrate that both spectroscopic techniques in combination with multivariate analysis are successful tools for the quantification of baicalin and total baicalein in Scutellariae radix, but it was found that ATR-IR spectroscopy provides higher accuracy in the given application. Furthermore it was proved that wild and cultivated samples can be distinguished by ATR-IR.

Simultaneous quantification of verbenalin and verbascoside in Verbena officinalis by ATR-IR and NIR spectroscopy.

Attenuated-total-reflectance infrared spectroscopy (ATR-IR) and near-infrared diffuse reflectance spectroscopy (NIR) in hyphenation with multivariate analysis was utilized to quantify verbenalin and verbascoside in Verbena officinalis. A new high performance liquid chromatography (HPLC) method as a reference was established and validated. For both vibrational spectroscopic methods test-set and cross validation were performed. Different data-pre-treatments like SNV, 1st and 2nd derivative were applied to remove systematic errors and were evaluated. Quality parameters obtained for the test-set validation revealed that ATR-IR (verbenalin: R(2)=0.94, RPD=4.23; verbascoside: R(2)=0.93, RPD=3.63) has advantages over NIR (verbenalin: R(2)=0.91, RPD=3.75; verbascoside: R(2)=0.80, RPD=2.35) in the given application.

Fourier transform infrared imaging analysis in discrimination studies of bladder cancer.

Bladder carcinoma represents more than 4% of all cancer diseases in Austria. The histomorphological evaluation is invasive and remains a subjective and time consuming technique. On account of this it is necessary to find novel non-invasive approaches which support the pathologists for histological recognition to identify malignancy at an early stage. In the present study, Fourier transform infrared (FTIR) microscopic imaging was combined with univariate and multivariate data analysis methods to study bladder carcinoma tissue sections in detail. The possibility to collect IR spectra of bladder carcinoma tissue sections employing an optimized analytical protocol is demonstrated. The correlation between FTIR microscopic imaging and the morphological tissue features obtained by histological staining of the sections demonstrated that many histomorphological tissue patterns can be visualized in the colour images. The routine generation of high quality imaging data is enabled because of the combination of FTIR technology and optimized sample preparation techniques. This opens a new quality of spectroscopic analyses of cancerous tissue, allowing exploration of molecular changes associated with the histopathological morphology.

Fourier transform infrared imaging analysis in discrimination studies of St. John's wort (Hypericum perforatum).

In the present study, Fourier transform infrared (FTIR) imaging and data analysis methods were combined to study morphological and molecular patterns of St. John's wort (Hypericum perforatum) in detail. For interpretation, FTIR imaging results were correlated with histological information gained from light microscopy (LM). Additionally, we tested several evaluation processes and optimized the methodology for use of complex FTIR microscopic images to monitor molecular patterns. It is demonstrated that the combination of the used spectroscopic method with LM enables a more distinct picture, concerning morphology and distribution of active ingredients, to be gained. We were able to obtain high-quality FTIR microscopic imaging results and to distinguish different tissue types with their chemical ingredients.

Fourier transform infrared imaging analysis in discrimination studies of squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC) of the oral cavity and oropharynx represents more than 95% of all malignant neoplasms in the oral cavity. Histomorphological evaluation of this cancer type is invasive and remains a time consuming and subjective technique. Therefore, novel approaches for histological recognition are necessary to identify malignancy at an early stage. Fourier transform infrared (FTIR) imaging has become an essential tool for the detection and characterization of the molecular components of biological processes, such as those responsible for the dynamic properties of tumor progression. FTIR imaging is a modern analytical technique enabling molecular imaging of a complex biological sample and is based on the absorption of IR radiation by vibrational transitions in covalent bonds. One major advantage of this technique is the acquisition of local molecular expression profiles, while maintaining the topographic integrity of the tissue and avoiding time-consuming extraction, purification, and separation steps. With this imaging technique, it is possible to obtain unique images of the spatial distribution of proteins, lipids, carbohydrates, cholesterols, nucleic acids, phospholipids, and small molecules with high spatial resolution. Analysis and visualization of FTIR imaging datasets are challenging and the use of chemometric tools is crucial in order to take advantage of the full measurement. Therefore, methodologies for this task based on the novel developed algorithm for multivariate image analysis (MIA) are often necessary. In the present study, FTIR imaging and data analysis methods were combined to optimize the tissue measurement mode after deparaffinization and subsequent data evaluation (univariate analysis and MIAs). We demonstrate that it is possible to collect excellent IR spectra from formalin-fixed paraffin-embedded (FFPE) tissue microarrays (TMAs) of OSCC tissue sections employing an optimised analytical protocol. The correlation of FTIR imaging to the morphological tissue features obtained by histological staining of the sections demonstrated that many histomorphological tissue patterns can be visualized in the colour images. The different algorithms used for MIAs of FTIR imaging data dramatically increased the information content of the IR images from squamous cell tissue sections. These findings indicate that intra-operative and surgical specimens of squamous cell carcinoma tissue can be characterized by FTIR imaging.

Selected Extracts of Chinese Herbal Medicines: Their Effect on NF-κB, PPARα and PPARγ and the Respective Bioactive Compounds.

Chinese herbal medicinal (CHM) extracts from fourteen plants were investigated in cell-based in vitro assays for their effect on nuclear factor κB (NF-κB), a key regulator of inflammation, as well as on peroxisome proliferator-activated receptors (PPARs) being key regulators of genes involved in lipid and glucose metabolism. 43% of the investigated CHMs showed NF-κB inhibitory and 50% PPARα and PPARγ activating effects. Apolar extracts from cortex and flos of Albizia julibrissin Durazz. and processed rhizomes of Arisaema sp. and Pinellia ternata (Thunb.) Breit. that effectively inhibited TNF-α-induced NF-κB activation and dose-dependently activated PPARα and PPARγ were further investigated. Bioassay-guided fractionation and analysis by GC-MS led to the identification of fatty acids as PPAR agonists, including linoleic and palmitic acid.

Near infrared spectroscopy patents for the physicochemical characterization of nanomaterials: the road from production to routine high-throughput quality control.

The measurement of the physical and chemical ("physicochemical") properties of nanomaterials used in industry and science including chemistry, pharmacy, medicine, toxicology, etc., is time-consuming, expensive and requires a lot of experience of a well trained lab staff. Near-infrared spectroscopy (NIR; 4.000-12.000 cm(-1)), working in the wavelength region with the highest IR energy, allows obtaining multifactorial information of the material under investigation due to the occurrence of a high number of combination and overtone vibrations. Coupling of an optimized and well-designed measurement technique with multivariate data analysis (MVA) leads to a non-destructive, fast, reliable and robust novel NIR technique for the fast and non-invasive physicochemical characterization, which is suitable for high-throughput quality control due to the short analyses times of only a few seconds. In the following chapters, the patented basic NIR techniques full-filling these aims are introduced, described, summarized and critically discussed.

Morphological and tissue characterization of the medicinal fungus Hericium coralloides by a structural and molecular imaging platform.

In this study the potential of new imaging techniques such as Magnetic Resonance Imaging (MRI), Matrix-Assisted Laser Desorption/Ionization (MALDI) profiling mass spectrometry ("MALDI Profiling") and Fourier Transform Infrared (FTIR) spectroscopic imaging was evaluated to study morphological and molecular patterns of the potential medicinal fungus Hericium coralloides. For interpretation, the MALDI profiling, FTIR imaging and MRI results were correlated with histological information gained from Scanning Electron Microscopy (SEM) and Light Microscopy (LM). Additionally we tested several evaluation processes and optimized the methodology for use of complex FTIR images to monitor molecular patterns. It is demonstrated that the combination of these spectroscopic methods enables to gain a more distinct picture concerning morphology and distribution of active ingredients. We were able to obtain high quality FTIR imaging and MALDI-profiling results and to distinguish different tissue types with their chemical ingredients. Beside this, we have created a 3-D reconstruction of a mature Hericium basidioma, based on the MRI dataset: analyses allowed, for the first time, a realistic approximation of the "evolutionary effectiveness" of this bizarrely formed basidioma type, concerning the investment of sterile tissue and its reproductive output (production of basidiospores).

Chemically modified diamond-like carbon (DLC) for protein enrichment and profiling by MALDI-MS.

The development of new high throughput methods based on different materials with chemical modifications for protein profiling of complex mixtures leads towards biomarkers; used particularly for early diagnosis of a disease. In this work, diamond-like carbon (DLC) is developed and optimized for serum protein profiling by matrix-assisted laser/desorption ionization mass spectrometry (MALDI-MS). This study is carried out in connection with a material-based approach, termed as material-enhanced laser desorption ionization mass spectrometry. DLC is selected as carrier surface which provides large surface to volume ratio and offers high sensitivity. DLC has a dual role of working as MALDI target while acting as an interface for protein profiling by specifically binding peptides and proteins out of serum samples. Serum constituents are bound through immobilized metal ion affinity chromatography (IMAC) functionality, created through glycidyl methacrylate polymerization under ultraviolet light followed by further derivatization with iminodiacetic acid and copper ion loading. Scanning electron microscopy highlights the morphological characteristics of DLC surface. It could be demonstrated that IMAC functionalized DLC coatings represent a powerful material in trapping biomolecules for their further analysis by MALDI-MS resulting in improved sensitivity, specificity and capacity in comparison to other protein-profiling methods.

GC-MS method for the simultaneous determination of ß-blockers, flavonoids, isoflavones and their metabolites in human urine.

A sensitive and selective method based on gas chromatography hyphenated to mass spectrometry (GC-MS) for the screening of 23 different compounds including ß-blockers, flavonoids, isoflavones and metabolites in human urine sample was developed and validated. The present paper reports, for the first time, the method for the simultaneous determination of ß-blockers, isoflavones, flavonoids and metabolites in human urine samples. When flavonoids are ingested in combination with drugs that have a narrow therapeutic range, interactions between flavonoids and drugs should be investigated. Substances of interest were extracted from urine samples by solid-phase extraction (SPE) employing a mixture of tert-butyl methyl ether:methanol:formic acid (4.5:4.5:1; v/v/v) as a mobile phase and Oasis HLB (Waters) as a stationary phase. Before extraction, urine samples were incubated with ß-glucuronidase/sulfatase in order to achieve enzymatic hydrolysis. Before GC-MS analysis the analytes had to be derivatized with N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) into their trimethylsilyl derivatives by incubating for 60 min at 60 °C. Statistical central composite design and response surface analysis were used to optimize the derivatization reagent. These multivariate procedures were efficient in determining the optimal separation condition, using peak areas as responses. The calibration curves were indicative of high linearity (r² ≥ 0.9992) in the range of interest for each analyte. LODs (S/N=3) ranged between 0.6 and 9.7 ng/ml. Intra-day and inter-day precision (CV, %) was less than 4.96%, accuracy between 0.01 and 4.98% and recovery was found in the range from 70.20 to 99.55%. The developed method can be applied to the routine determination of examined compounds' concentrations in human urine. Moreover the method is suitable for detecting pharmaceutical compounds containing ß-blockers, isoflavones and flavonoids in urine after administration to humans.

Protein profiling for cancer biomarker discovery using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and infrared imaging: a review.

Cancer biomarker refers to a substance or process that is indicative of the presence of cancer in the body. A biomarker might be either a molecule secreted by a tumor or it can be a specific response of the body to the presence of cancer. Cancer biomarker-based diagnostics have applications for establishing disease predisposition, early detection, cancer staging, therapy selection, identifying whether or not a cancer is metastatic, therapy monitoring, assessing prognosis, and advances in the adjuvant setting. Full adoption of cancer biomarkers in the clinic has to date been slow, and only a limited number of cancer biomarker products are currently in routine use. Among proteomic technologies, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) is a technique that has allowed rapid progress in cancer biology. Different further developed methods including e.g. SELDI (surface-enhanced laser desorption/ionization) and MELDI (material-enhanced laser desorption/ionization) are simple and high-throughput techniques that analyze with high sensitivity and specificity intact proteins expressed in complex biological mixtures, such as serum, urine, and tissues. The combination of mass spectrometry (MS) with infrared (IR) spectroscopic imaging is an attempt to combine different technologies in systems analytics. Both MALDI-TOF and infrared tissue imaging enable studying proteins distribution in tissue samples with a resolution down to 50 and 5 µm, respectively. In this review, we summarize recent applications and the synergistic combination of these new technologies to proteomic profiling for cancer biomarker discovery.

Near-infrared reflection spectroscopy (NIRS) as a successful tool for simultaneous identification and particle size determination of amoxicillin trihydrate.

A successful application of NIR spectroscopy (NIRS) in combination with multivariate data analysis (MVA) for the simultaneous identification and particle size determination of amoxicillin trihydrate particles was developed. Particle size analysis was ascertained by NIRS in diffuse reflection mode on different particle size fractions of amoxicillin trihydrate with D90 particle diameters ranging from 6.9 to 21.7 µm. The present problem of fractionating the powder into good enough size fractions to achieve a stable calibration model was solved. By probing dried suspensions measurement parameters were optimized and further combined with the best suitable chemometric operations. Thereby the quality of established regression models could be improved considerably. A linear coherence between particle size and absorbance signal was found at specific wavenumbers. Satisfactory clustering by particle size was achieved by principal component analysis (PCA) whereas partial least squares regression (PLSR) and principal component regression (PCR) was compared for quantitatively calibrating the NIRS data. PLSR turned out to predict unknown test samples slightly better than PCR.

Use of Chromatographic (SPE-HPLC) and Spectrophotometric Methods for Differentiation of Salix Species Through Correlation Analysis and FreeViz Projection.

The major concern of the present article is research into the combination of analytical assessment and multi-correlative data interpretation. For this purpose, a high performance liquid chromatography (HPLC) procedure was developed for the simultaneous quantification of salicin, salicylalcohol derivatives, phenolic acids, flavonoids, and monomeric catechins after solid phase extraction (SPE). On the basis of an established and validated HPLC method, 49 different Salix samples were extracted, purified, and analysed. Furthermore, the quantity of total polyphenols (Folin-Ciocalteau colorimetric reaction) and the antioxidant activity (DPPH radical scavenging activity test) were determined and correlated. This yielded correlation coefficients at P-values less than 0.05 of 0.775, 0.967, 0.932, and 0.989 for Salix fragilis, Salix rubens, Salix purpurea (2006), and Salix purpurea (2007), respectively. Correlation with mean values of each species between total polyphenols content and % DPPH inhibition values occurred at a correlation coefficient (r) of 0.851. Linear correlations of quantified HPLC data with DPPH data and with total polyphenols content could also be found for salicin, gentisic acid, naringin, and salicylic acid. Finally, by combining HPLC data with total polyphenols content and antioxidant capacity through 3-D scatter plots and FreeViz data projection, it was shown that primarily the amount of epicatechin and saligenin beside DPPH values and total polyphenols content enable the classification into plant species and further by year of harvest.

Infrared-spectroscopy: a non-invasive tool for medical diagnostics and drug analysis.

Constant development enabled Infrared (IR) spectroscopy to become a widely used, non-invasive tool for fast sample analyses with less to no pre-preparation. Furthermore, computational data handling is no more a limiting factor and hence, IR measurements are predestined for clinical diagnostics and drug analysis. Within this review the focus was put on clinical topics of high interest. One example is Alzheimer's disease, where the exact metabolism is still not clarified, or blood glucose monitoring for high throughput screening of patients without taking any drop of blood. The second section of this manuscript was focused on the analysis of drugs. The detection of physico-chemical parameters in pharmaceutics and the improvement of industrial proceedings allowed a dramatic increase of quality of produced medicine. In pharmaceutical industries problems with the equable allocation of agents occurs especially in scaling up processes. IR-analyzing-techniques serve as fast and precise indicators for the detection of active components and their distribution in tablets. In combination with statistical factors and medical investigations pharmaceuticals can be improved from their development until their application, and every step can be easily controlled by IR spectroscopy.

Near-infrared spectroscopic study on guest-host interactions among G0-G7 amine-terminated poly(amidoamine) dendrimers and porous silica materials for simultaneously determining the molecular weight and particle diameter by multivariate calibration techniques.

The guest-host interactions of poly(amidoamine) (PAMAM) dendrimers and porous silica surfaces were investigated by near-infrared (NIR) diffuse reflection spectroscopy. G0-G7 of amine-terminated PAMAM (PAMAM-NH2) dendrimers were analyzed comprising early, mid, and late generations. For early stages, the adsorption process of the partly protonated dendrimers to the negatively charged silica surface strongly depends on the size/shape characteristics of the guest (PAMAM-NH2 dendrimers) and host (porous silica) materials. G0-G4 (15-45 A) show smaller particle sizes than the pore diameter of the silica (60 A) and thus have access to the interior surface of the host material. For mid and later stages (G5-G7; 54-81 A) only low amounts of the dendrimers adsorb to the silica surface due to the inaccessibility to the interior surface. The loading capacity of the silica material with adsorbed PAMAM-NH(2) was evaluated by means of capillary zone electrophoresis (CZE), whereas deviations from the theoretical to the effective particle size and molecular weight (MW) was determined by gas-phase electrophoretic mobility molecular analysis (GEMMA) and matrix-assisted laser desorption/ionization linear time-of-flight mass spectrometry (MALDI-lin TOF-MS). Deviations from the theoretical to the actual values showed a maximum of 13.8% and 28.0% for the particle size and MW, respectively. The NIR absorption spectra show a distinct band at 4932 cm(-1) (nu(sym) (NH) + amide II) due to the adsorbed dendrimers. It was found that the absorbance tends to increase with decreasing generation number. On this basis multivariate calibration was performed with the theoretical data and the data obtained by GEMMA and MALDI-lin TOF-MS. All in all, the calculated partial least-squares regression (PLSR) model containing the GEMMA/MALDI-lin TOF-MS reference values showed better results than the models exclusively calculated from the theoretical values. This indicates that the theoretical values do not imply the structural imperfections arising during the synthesis that may be present in the PAMAM-NH2 dendrimers.

Development and application of Fourier-transform infrared chemical imaging of tumour in human tissue.

Fourier-transform infrared (FT-IR) based mapping and imaging is a fast emerging technology which is being increasingly applied to investigate tissues in the high-throughput mode. The high resolution close to the cellular level, the possibility to determine the bio-distribution of molecules of interest (proteins, peptides, lipids, carbohydrates) without any pre-treatment and the offer to yield molecular structure information have brought evidence that this technique allows to gain new insights in cancer pathology. Thus, several individual mainly protein and peptide cancer markers ("biomarkers") can be identified from FT-IR tissue images, enabling accurate discrimination between healthy and tumour areas. Optimal data acquisition (spatial resolution, spectral resolution, signal to noise ratio), classification, and validation are necessary to establish practical protocols that can be translated to the qualitative and quantitative clinical routine analysis. Thereby, the development of modern fast infrared imaging systems has strongly supported its acceptance in clinical histopathology. In this review, the necessity of analysis based on global cancer statistics, instrumental setups and developments, experimental state of the art are summarised and applications to investigate different kinds of cancer (e.g., prostate, breast, cervical, colon, oral cavity) are shown and discussed in detail.

Simultaneous determination of the micro-, meso-, and macropore size fractions of porous polymers by a combined use of Fourier transform near-infrared diffuse reflection spectroscopy and multivariate techniques.

Fourier transform near-infrared (FT-NIR) diffuse reflection spectroscopy was used in combination with principal component analysis and partial least-squares regression to simultaneously determine the physical and the chemical parameters of a porous poly(p-methylstyrene-co-1,2-bis(p-vinylphenyl)ethane) (MS/BVPE) monolithic polymer. Chemical variations during the synthesis of the polymer material can alter the pore volume and pore area distributions within the polymer scaffold. Furthermore, mid-infrared and near-infrared (NIR) spectroscopic chemical imaging was implemented as a tool to assess the uniformity of the samples. The presented study summarizes the comparative results derived from the spectral FT-NIR data combined with chemometric techniques. The relevance of the interrelation of physical and chemical parameters is highlighted whereas the amount of MS/BVPE (%, v/v) and the quantity (%) of micropores (diameter, d < 6 nm), mesopores (6 nm < d < 50 nm), and macropores (50 nm < d < 200 nm) could be determined with one measurement. For comparison of the quantitative data, the standard error of prediction (SEP) was used. The SEP for determining the MS/BVPE amount in the samples showed 0.35%, for pore volume quantiles 1.42-8.44%, and for pore area quantiles 0.38-1.45%, respectively. The implication of these results is that FT-NIR spectroscopy is a suitable technique for the screening of samples with varying physicochemical properties and to quantitatively determine the parameters simultaneously within a few seconds.

Characterisation of different types of hay by solid-phase micro-extraction-gas chromatography mass spectrometry and multivariate data analysis.

The volatile organic compounds of different ground hay samples from Austria, Italy and Switzerland were collected at 50 degrees C on a Supelco Carbowax Divinylbenzene headspace solid-phase microextraction fibre, separated by capillary gas chromatography on an HP5-ms column running a temperature programme and using helium as carrier gas, detected with a mass sensitive detector and studied with principal component analyses after autoscaling selected variables. The analytes, mainly mono- and sesquiterpenes, were able to cluster differences resulting from the site of production. Coumarin can be used to differentiate hay grown north and south of the main chain of the Alps. Acetic acid is appropriate for distinguishing between hay from Kastelruth and Passeier Valley, two South Tyrolean regions. The average linalool content in aftermath is higher than in hay.

Quantitative and qualitative investigation of the main flavonoids in heartsease (Viola tricolor L.).

Liquid chromatography coupled to electrospray ionization tandem mass spectrometry (MSn) is used for the analysis of flavonoids in heartsease (Viola tricolor L.). Our data suggested that the two main flavonoid components were violanthin (6-C-glucosyl-8-C-rhamnosyl apigenin) and rutin (3-O-rutinosyl quercetin). The identification of rutin was confirmed by comparing its retention time, UV spectrum, molecular mass, and fragmentation pattern with the reference standard. In this paper, we also report on the quantitative analysis of rutin by high-performance liquid chromatography. According to our results, heartsease herb contained 420+/-1.17 microg/g rutin.

Nano-structured support materials, their characterisation and serum protein profiling through MALDI/TOF-MS.

In the bioanalytical era, novel nano-materials for the selective extraction, pre-concentration and purification of biomolecules prior to analysis are vital. Their application as affinity binding in this regard is needed to be authentic. We report here the comparative application of derivatised materials and surfaces on the basis of nano-crystalline diamond, carbon nanotubes and fullerenes for the analysis of marker peptides and proteins by material enhanced laser desorption ionisation mass spectrometry MELDI-MS. In this particular work, the emphasis is placed on the derivatization, termed as immobilised metal affinity chromatography (IMAC), with three different support materials, to show the effectiveness of MELDI technique. For the physicochemical characterisation of the phases, near infrared reflectance spectroscopy (NIRS) is used, which is a well-established method within the analytical chemistry, covering a wide range of applications. NIRS enables differentiation between silica materials and different fullerenes derivatives, in a 3-dimensional factor-plot, depending on their derivatizations and physical characteristics. The method offers a physicochemical quantitative description in the nano-scale level of particle size, specific surface area, pore diameter, pore porosity, pore volume and total porosity with high linearity and improved precision. The measurement takes only a few seconds while high sample throughput is guaranteed.